Characterisation of a metalloendopeptidase (PepO) from Streptococcus pyogenes

D Ngu1, S Williams2 and B Kobe1

  1. School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
  2. Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), are Gram-positive, β-haemolytic human pathogens that are able to cause infections ranging from the acute (e.g. bacterial pharyngitis, impetigo) to the severe (e.g. streptococcal toxic shock syndrome, necrotising fasciitis). Their significant impact on society makes it crucial to study the virulence factors that contribute to their virulence. Recently, PepO, a predicted zinc-binding metalloendopeptidase with sequence similarity to the M13 peptidase family, has been implicated as a virulence factor. Previous studies of PepO from other streptococcal species have found that it inhibits the classical complement pathway and binds to the C1q complement protein, plasminogen and fibronectin. In GAS, PepO is found to be a quorum quencher that mediates intercellular signalling through the degradation of active short peptide pheromones of the Rgg2/3 pathway and its expression is regulated by the CovRS system. In this study, I aimed to structurally characterise PepO both as an apoenzyme and a holoenzyme with the zinc (II) metal ion cofactor. PepO was expressed as fusion proteins with N-terminal affinity tags and purified via affinity chromatography and size exclusion chromatography. Experimental analyses suggested that PepO underwent proteolysis during the purification process and the addition of a metalloprotease inhibitor, 1,10-phenanthroline, prevented further degradation. Based on these results, I hypothesised that autocatalytic proteolysis may be a novel regulatory function of PepO. Future work include further optimisation of the purification conditions and crystallisation of PepO in both active and inactive forms for structural characterisation.